Background-Cyclic guanosine monophosphate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides, such as atrial or brain natriuretic peptide, which activate the soluble and particulate forms of guanylyl cyclase, respectively. However, natriuretic peptides and NO donors exert different effects on cardiac and vascular smooth muscle function. We therefore tested whether these differences are due to an intracellular compartmentation of cGMP and evaluated the role of phosphodiesterase (PDE) subtypes in this process. Methods and Results-Subsarcolemmal cGMP signals were monitored in adult rat cardiomyocytes by expression of the rat olfactory cyclic nucleotide-gated (CNG) channel ␣-subunit and recording of the associated cGMP-gated current (I CNG ). Atrial natriuretic peptide (10 nmol/L) or brain natriuretic peptide (10 nmol/L) induced a clear activation of I CNG , whereas NO donors (S-nitroso-N-acetyl-penicillamine, diethylamine NONOate, 3-morpholinosydnonimine, and spermine NO, all at 100 mol/L) had little effect. The I CNG current was strongly potentiated by nonselective PDE inhibition with isobutyl methylxanthine (100 mol/L) and by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (10 mol/L) and Bay 60-7550 (50 nmol/L). Surprisingly, sildenafil, a PDE5 inhibitor, produced a dose-dependent increase of I CNG activated by NO donors but had no effect (at 100 nmol/L) on the current elicited by atrial natriuretic peptide. Conclusions-These results indicate that in rat cardiomyocytes (1) the particulate cGMP pool is readily accessible at the plasma membrane, whereas the soluble pool is not; and (2) PDE5 controls the soluble but not the particulate pool, whereas the latter is under the exclusive control of PDE2. Differential spatiotemporal distributions of cGMP may therefore contribute to the specific effects of natriuretic peptides and NO donors on cardiac function. Key Words: cyclic GMP Ⅲ natriuretic peptides Ⅲ nitric oxide Ⅲ phosphodiesterases Ⅲ sildenafil C yclic guanosine monophosphate (cGMP) is a ubiquitous intracellular second messenger in the cardiovascular system. In the heart, acute elevation of cGMP concentration usually exerts negative metabolic as well as inotropic effects, 1,2 whereas chronic elevation prevents and reverses cardiac hypertrophy. 3-5 cGMP synthesis is controlled by 2 types of guanylyl cyclases (GC) that differ in their cellular location and activation by specific ligands: a particulate GC (pGC) present at the plasma membrane, which is activated by natriuretic peptides such as atrial (ANP), brain (BNP), and C-type natriuretic peptide 6 -8 ; and a soluble guanylyl cyclase (sGC) present in the cytosol and activated by nitric oxide (NO). 8,9 Clinical Perspective p 2228Although NO and natriuretic peptides use cGMP as a common second messenger, there are many instances in which activation of pGC and sGC leads to different functional effects. 10 -14 One explanation for these divergent effects is that cGMP rises in specific subcellular...

1 The e ects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I Ca ) and intracellular cyclic AMP concentration ([cAMP] i ) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the di erent cardiac PDE subtypes was con®rmed by RT ± PCR. 2 IBMX (100 mM), a broad-spectrum PDE inhibitor, increased basal I Ca by 120% and [cAMP] i by 70%, similarly to a saturating concentration of the b-adrenoceptor agonist isoprenaline (1 mM). However, MIMX (1 mM), a PDE1 inhibitor, EHNA (10 mM), a PDE2 inhibitor, cilostamide (0.1 mM), a PDE3 inhibitor, or Ro 20-1724 (0.1 mM), a PDE4 inhibitor, had no e ect on basal I Ca and little stimulatory e ects on [cAMP] i (20 ± 30%). 3 Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any e ect on I Ca or [cAMP] i . While all combinations tested signi®cantly increased [cAMP] i (40 ± 50%), only cilostamide (0.1 mM)+Ro20-1724 (0.1 mM) produced a signi®cant stimulation of I Ca (50%). Addition of EHNA (10 mM) to this mix increased I Ca to 110% and [cAMP] i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 mM) or isoprenaline (1 mM). 4 When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I Ca by (&40% and had negligible e ect on [cAMP] i , each selective PDE inhibitor induced a clear stimulation of [cAMP] i and an additional increase in I Ca . Maximal e ects on I Ca were &8% for MIMX (3 mM), &20% for EHNA (1 ± 3 mM), &30% for cilostamide (0.3 ± 1 mM) and &50% for Ro20-1724 (0.1 mM). 5 Our results demonstrate that PDE1-4 subtypes regulate I Ca in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I Ca , all four PDE subtypes determine the response of I Ca to a stimulus activating cyclic AMP production, with the rank order of potency PDE44PDE34PDE24PDE1.